Biokinetics of engineered nano-TiO₂ in rats administered by different exposure routes: implications for human health

Commentary

Over the past decade, nanotechnology has matured from an emerging discipline to a mainstream technology with hundreds of high-volume materials, products, or high-frequency applications currently on the market. Nanotoxicology, the discipline that came into existence primarily because of challenges posed by nanotechnology, has also evolved over this past decade into a mature, interdisciplinary science. Several challenges were posed to the nanotoxicology community a decade ago: the large number of ENM, each potentially being manufactured with an overwhelming number of variations in their size and other physico-chemical properties; novel behavior of materials at the nanoscale; inadequacy of existing mass-based dose metrics and the need for new exposure and dose metrics, to name a few. Perhaps the greatest concern, in our opinion, was the ability of nanomaterials to cross biological barriers: the skin, the GI tract, and the gas exchange region-and to enter the systemic circulation, from wherein they would gain access to other organs – the heart, the brain, the liver, and immune system, to name a few. The scope of nanoparticle toxicology appeared then to expand greatly beyond conventional particle toxicology. This concern posed a unique technical challenge: how to trace and quantify nanoparticles in complex biological media and living organisms?

Such technical and conceptual challenges demanded innovations in the testing approaches and the test systems. Major efforts were dedicated for developing high-throughput in vitro testing platforms and to expanding testing endpoints to include more complex systems and pathways, as an alternative strategy to animal testing (Nel, 2013; Nel et al., 2013; Zhang et al., 2012). Animal inhalation studies using realistic nanoparticle aerosols presented several technical and economic challenges. This was particularly true for some classes of nanomaterials, such as CNTs, which in the early days were priced at $1000 or more per gram. Long-term inhalation studies – the ones most relevant to answering some of the more fundamental nanotoxicology questions were particularly difficult to conduct. Intratracheal instillation was developed as an alternative delivery technique for lung inhalation exposures. A quick PubMed search using terms “intratracheal instillation and nanomaterials” produced 260 hits, with all publications but 5 published in the last decade. Although the first report of IT dates back to 1997 and corresponds to fullerenes, the technique really took off in 2006–2007. Intravenous injection (IV) of nanoparticle dispersions directly into blood stream was being exploited in nanomedicine for drug delivery and imaging applications and adopted in nanotoxicology for biokinetics studies. A quick search in PubMed using the term “intravenous injection and nanoparticles” yielded over 1700 hits, dating as far back as 1982. Surprisingly, the first PubMed report assessing toxicity of doxorubicin loaded on polyisobutylcyanoacrylate nanoparticles following IV infusion in mice was published in the early 1980s (Couvreur et al., 1982). The term “nanoparticle” was used here, rather than the “ultrafine particle” found in the literature of that time. The published literature started to grow exponentially after 2005–2006, with an average of ∼220 publications/year in 2015, upto 10× from a decade earlier. Half of this literature was published during the last 5 years.

Of course, the bulk of nanotoxicity testing during this time period happened using in vitro methods, i.e., assessing effects on cell lines or cell co-cultures. The picture here is similar to IT and IV. Over 10,300 hits were generated in PubMed using the term “nanoparticle and cell toxicity”. The exponential growth of in vitro testing of nanoparticles started in 2006. The number of papers/year (a crude metric of research activity) increased 10-fold over the past decade, from an average of 147 to over 1500 publications/year. The issue of assay validation for nanotoxicology research – both with regard to the established assays (starting with cell viability); and even more critically of new assays and techniques – were cited frequently in the published literature and in scientific meetings, but did not gain traction for a number of years. As an example, one of the first challenges for nanotoxicology research, perhaps the very first one, and the simplest conceptual problem, was dispersion of ENM in a relevant media. How should nanomaterials be dispersed? In what dispersion media? Does this matter and if it does, how does it affect the nanotoxicology testing outcomes and, of course, why? These factors have had a significant impact in vitro (DeLoid et al., 2015, 2017; Pal et al., 2015; Watson et al., 2016). More recent comparative work has shown that IT dose rate and mode of administration impact considerably the study outcomes (Baisch et al., 2014; Silva et al., 2014). What effects did such dispersions have during their subsequent use in IT and IV applications? This has been harder to settle.

Documenting quantitatively the biokinetics of nanoparticle uptake and translocation was easier said than done. Tracking and visualizing individual nanoparticles in complex and dynamic biological systems, especially in live organisms, remains a daunting task today. Translocation of ENM from the lungs seems to happen at low rates in general (<0.001–1%) (Geiser & Kreyling, 2010; Geiser et al., 2014; Moller et al., 2008; Scheuch et al., 2008), much smaller than the earliest translocation values (Nemmar et al., 2002). It is not our intention to criticize any of the mentioned studies, but rather to remind ourselves that for a combination of reasons (lack of metrology technology, lack of experience working with ENM, enthusiasm of early discoveries, good faith assumptions, etc.,), high-quality studies on nanoparticle biokinetics in animals and especially in humans have been difficult to produce. In several occasions as mentioned above, the basic assumptions turned out to be wrong, and a substantial amount of published nanotoxicology research findings (as it relates to lack of in vitro dosimetry, unrealistically high dose rates in IT studies, interferences on spectrophotometric assays, etc.) may not be reproducible, and consequently, of little scientific value!

In this issue of Nanotoxicology, we have another significant discovery by Kreyling et al.: that the intravenous injection method for ENMs does not represent a suitable surrogate biokinetic approach for making accurate assumptions regarding particle exposure, uptake or retention following oral or pulmonary routes of exposures. Accordingly, the authors conclude that IV infusion of ENM may have little methodological validity for biokinetic studies and nanotoxicology research!

In a series of three publications, Kreyling et al. investigated in great details the biokinetics and translocation of a single reasonable dose of nano-TiO₂ in rats, administered via three main pathways: IV, IT and gavage (Kreyling et al., 2017a, b, c). The authors employed radiolabeled TiO₂ with ⁴⁸V (⁴⁸V-nTiO_2, 70 nm median primary size), which enabled the authors to follow the kinetics of translocation and the overall biodistribution of ⁴⁸V-nTiO₂ in various tissues in great detail over 28 day post-exposure and to conduct detailed mass balance estimates. Other important methodological aspects of these studies include the use of the same ⁴⁸V-nTiO₂ material, unified approaches for nanoparticle tracking and mass balance calculations, enabling cross-comparison between the three studies, and extensive corrections for possible ⁴⁸V radiolabel leaching. The use of ⁴⁸V gamma-emitting radionuclide as a label on nTiO₂, enables high-sensitivity detection over a broad concentration range of five orders of magnitude, free of interferences from background Ti originating from dietary and environmental exposures of the animals. This series of studies provides among the highest quality biokinetics data available to date. These studies represent “state of the art” approaches, and are the first to assess biokinetics of poorly soluble nanoparticles by all of the three most common routes of exposure. It is this combination of features – meticulous attention to experimental details, excellent quality experimental data, and the simultaneous investigation of the three exposure routes for an important commercial nanoparticle – that makes this series of studies particularly noteworthy and, they will likely become a landmark set of studies on nanoparticle biokinetics. In this context, we highlight some of the most noteworthy findings from these studies and their implications regarding fate and clearance of nanoparticles following various exposure pathways, and the underlying mechanisms driving such findings.

In their trilogy, the authors report interesting translocation and clearance data for nTiO₂. Especially insightful are the data related to (i) relocation of nanoparticles within the lungs - internalization by various cell types (epithelial cells and alveolar macrophages), relocation from the lung surface into the epithelium and interstitium, and their reappearance into the alveolar space; (ii) contribution of lung particle clearance mechanisms, such as the (relatively fast) mucociliary escalator (MC) and (slower) alveolar macrophage clearance, to the GI tract dose following swallowing of MC cleared nanoparticles; (iv) translocation of a small fraction of ingested NP from the GI tract into the systemic circulation, including the relative contribution of swallowed nanoparticles originating from the lungs; and retention and accumulation of NP into several secondary organs 28 days post-exposure, most notably the liver and spleen. The translocation kinetics of nTiO₂ following IT and GI routes were relatively similar to each other, but rather distinct form the IV route of administration.

However, we would like to emphasize one particular finding related to oral exposure of nTiO₂ and its translocation across the gastro-intestinal barrier, for the reason that ingestion of nanoparticles in general and nTiO₂ in particular, has received much attention in the nanotoxicology community recently. Following oral gavage (GI), ∼0.6% of ⁴⁸V–nTiO₂ was translocated to the blood stream in 1 h. The single dose was cleared by day 7, with ∼99.7% of the dose cleared in feces. However, the ∼0.3% retained dose did not clear from the body for at least 7 days. The nanomaterial showed up in the liver and continued to accumulate in the liver and spleen over the study period, a phenomenon related to these organs’ high capacity for nanoparticle uptake/sequestration via their mononucleated-phagocytic-system. Consistent with earlier hypotheses and data (Kreyling et al., 2002; Oberdorster et al., 2005), the relatively small fraction of nanoparticles that enter systemic circulation (from the lungs or the GI tract) are being scavenged by and retained in the liver. Although the translocated dose to the liver was 0.03% of the initial GI dose at 4 h (0.006% on day 7), long-term accumulation of nanoparticles in secondary organs under chronic exposures can be significant.

Implications

What does this mean for human health? Before we delve into this question, we would like to remind ourselves that these studies were conducted in rats and that, because of significant differences in the GI tract physiology and size, the findings cannot be transferred directly to humans. In this regard, it is known that the biokinetics/particle distribution patterns of inhaled particulates are very different when comparing rats to humans. In humans and monkeys, a significant majority (>80%) of inhaled particles deposit on alveolar surfaces or respiratory bronchioles translocate across epithelial cells to interstitial sites. In contrast, in rats the vast majority of particles which deposit in the lower respiratory tract remain within alveolar ducts, both phagocytized in alveolar macrophages and along alveolar epithelial cell surfaces (Nikula et al., 1997). This difference in interspecies particle distribution patterns (rat vs. human) results in disparities in pulmonary clearance rates of ∼60 days in rats vs. ∼400 days in humans (Gregoratto et al., 2010).

Nano-TiO₂ is an important commercial material, with thousands of tons produced annually, although it comprises only  <1% of the volume of pigment-grade TiO₂ particles. Pigment grade TiO₂ (size range ∼150–400 nm) is used primarily as a whitening agent due to its light scattering effects in numerous consumer products (paints, coatings, paper, textile, plastics), as well as a food additive in candies, sweets, chewing gums, diary creamers and other dairy products, beverages, pharmaceuticals (as a filler), and toothpastes. Nano-TiO₂ is used primarily in sunscreens, cosmetics, and catalytic applications. Limited surveys have found that many foods contained 0.01–1 mg TiO₂/serving (maximum 300 mg TiO₂/serving) (Lomer et al., 2000; Weir et al., 2012). Estimated human daily consumption based on limited studies varies widely from ∼0.2 to ∼200 μg TiO₂/kg body weight/day, with a maximum of 4 mg/kg body weight per day (Rompelberg et al., 2016; Weir et al., 2012). An adult may consume several milligrams of pigmented TiO₂ in a day. Of note, the food-grade TiO₂ (E171) used in these products contains less than 20% nanoscale particles, which disperse readily to form stable colloids. Therefore, ingestion of food and food-additives could be a significant exposure pathway for nano-TiO₂ in humans.

Apart from the nanoscale fraction, the rest of the pigment-grade TiO₂ component is much larger than industrial nano-TiO₂. What effect does this size difference have on particle clearance, retention, and translocation to extrapulmonary organs? The current set of studies do not address this issue directly, but comparative analysis with other similar studies on other types of nanoparticles by the authors suggests that translocation across biological barriers is smaller for larger particles (particularly true for nanoparticles of the same chemistry). Another important consideration is that, in spite of its high sensitivity, gamma spectroscopy cannot accurately document the cell-type interactions of nTiO₂ within an organ. Where exactly do these nanoparticles reside inside an organ? How does this storage/compartmentalization affect organ function and toxicity?

In addition to frequent ingestion of (primarily of pigment-grade TiO₂), additional exposure of particles to the gastrointestinal tract may occur due to mucociliary escalator clearance in occupational settings wherein TiO₂ production and processing occurs for which inhalation and hand-to-mouth contact are primary exposure pathways. Human chronic exposure via inhalation – is mostly occupational, but occasional consumer exposures, may further contribute to systemic inhalation and ingestion of pigment-grade TiO₂ particles. In light of chronic human exposures to (industrial nanoscale and pigment-grade) TiO₂ and evidence of limited translocation to systemic circulation and subsequent accumulation in the liver and spleen, it is reasonable to suggest that the nanotoxicology community should be attentive to possible long-term effects of such nanoparticle accumulation in the liver and possibly other organs; although it should be noted that the reticuloendothelial system (RES) is operative in the major organs (e.g., Kupffer cells in the liver). The challenge will continue to be the notable lack of nanoparticle biokinetics data in humans, especially in the extrapulmonary organs, which has been propagated in part by the ongoing technological and ethical difficulties of imaging and acquiring nanoparticle concentration data in various secondary organs. These data are critical for a realistic assessment of the long-term risks to humans under chronic exposures (Heringa et al., 2016). Future studies will certainly answer these questions!